Abstract

Annular apodization of the illumination and/or imaging pupils of an adaptive optics scanning light ophthalmoscope (AOSLO) for improving transverse resolution was evaluated using three different normalized inner radii (0.26, 0.39 and 0.52). In vivo imaging of the human photoreceptor mosaic at 0.5 and 10° from fixation indicates that the use of an annular illumination pupil and a circular imaging pupil provides the most benefit of all configurations when using a one Airy disk diameter pinhole, in agreement with the paraxial confocal microscopy theory. Annular illumination pupils with 0.26 and 0.39 normalized inner radii performed best in terms of the narrowing of the autocorrelation central lobe (between 7 and 12%), and the increase in manual and automated photoreceptor counts (8 to 20% more cones and 11 to 29% more rods). It was observed that the use of annular pupils with large inner radii can result in multi-modal cone photoreceptor intensity profiles. The effect of the annular masks on the average photoreceptor intensity is consistent with the Stiles-Crawford effect (SCE). This indicates that combinations of images of the same photoreceptors with different apodization configurations and/or annular masks can be used to distinguish cones from rods, even when the former have complex multi-modal intensity profiles. In addition to narrowing the point spread function transversally, the use of annular apodizing masks also elongates it axially, a fact that can be used for extending the depth of focus of techniques such as adaptive optics optical coherence tomography (AOOCT). Finally, the positive results from this work suggest that annular pupil apodization could be used in refractive or catadioptric adaptive optics ophthalmoscopes to mitigate undesired back-reflections.

Transverse resolution (left), axial resolution (center) and energy detected (right) in a confocal scanning instrument, as a function of the detector size for various relative pupil obscuration radii (ε), in terms of Airy disk radii (A.R.). The energy detected plots were generated assuming identical number of photons reaching the sample.

MTF of a confocal microscope with different apodizing binary mask combinations (one per row) with different radii (indicated by color) and pinhole sizes (one per column). These plots were calculated assuming 680 nm light, an eye with 17 mm focal length imaged through a 7.75 mm pupil diameter. The cut off spatial frequency (fmax) for an incoherent imaging system with uniform illumination (i.e. a flood illumination fundus camera) is provided for reference.

Registered averages of foveal cone photoreceptor AOSLO images. Each column corresponds to a different apodization mask normalized radius ε, while each row corresponds to a different apodization configuration. For each ε all images show the same retinal location. Image contrast has been stretched for display purposes only. Scale bar is 10μm across.

The top row shows the radial spectra of the foveal cone photoreceptor images in Fig. 4, with each column corresponding to a different apodizing mask inner radius ε, normalized to unit amplitude at zero frequency. The second row shows the ratios of the normalized spectra from images acquired with annular pupil(s) to the ones with full circular pupils. The cutoff spatial frequency for a non-scanning incoherent imaging system and the peak frequency associated to the cones are indicated for reference.

Registered averages of photoreceptor AOSLO images at 10° temporal from fixation. Each column corresponds to a different apodization mask normalized radius ε, while each row corresponds to a different apodization configuration. For each ε all images show the same retinal location. Image contrast has been stretched for display purposes only. Scale bar is 10μm across.

Radial spectra of the rod-dominated images in Fig. 6, with each column representing a different ε. The second row shows ratios of the normalized spectra from images taken with annular pupil(s) to the spectra of images taken with circular pupils.

Metrics

Table 1

Normalized autocorrelation central lobe FWHM changes due to pupil apodization corresponding to images in Fig. 4. Negative values indicate FWHM reduction that can be interpreted as sharpening of the image features.

Table 3

Cone counts for the images in Fig. 4. The manual counts are averages (and standard deviation) from 6 counts by 3 different observers.

ε = 0.26

ε = 0.39

ε = 0.52

Pupil masks

Automated

Manual

Automated

Manual

Automated

Manual

None

283

286 (1.3)

266

270 (1.5)

253

266 (2.1)

Illumination

306

309 (1.6)

318

322 (2.2)

380

385 (2.7)

Imaging

283

287 (2.3)

268

273 (1.3)

270

276 (2.6)

Both

301

306 (1.5)

323

329 (1.8)

350

354 (1.9)

Table 4

Normalized autocorrelation central lobe FWHM changes due to pupil apodization corresponding to images in Fig. 6. Negative values indicate FWHM reduction, that can be interpreted as sharpening of the image features.

Pupil masks

ε = 0.26 (%)

ε = 0.39 (%)

ε = 0.52 (%)

None

0.00

0.00

0.00

Illumination

−8.0

−12.7

−7.4

Imaging

−3.7

−2.2

3.3

Both

−11.2

−10.5

3.1

Table 5

Measured relative average photoreceptor intensities for images collected with constant PMT gain compared against predicted values (in parenthesis) corresponding to Fig. 6. The values used to describe the SCE are the best fit to our experimental data (ρcones = 0.15 mm−2 and ρrods = 0.11 mm−2) assuming a Gaussian intensity profile at the illumination pupil (σ = 2.9 mm) and a one Airy disk confocal aperture.

ε = 0.26

ε = 0.39

ε = 0.52

Pupil masks

Cones

Rods

Cones

Rods

Cones

Rods

None

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

Illumination

0.48 (0.68)

0.55 (0.73)

0.46 (0.42)

0.57 (0.49)

0.15 (0.21)

0.20 (0.27)

Imaging

0.59 (0.59)

0.72 (0.64)

0.33 (0.30)

0.36 (0.37)

0.11 (0.12)

0.13 (0.17)

Both

0.26 (0.40)

0.35 (0.47)

0.15 (0.13)

0.22 (0.18)

0.02 (0.03)

0.04 (0.05)

Table 6

Ratios of the average image intensity from cones to that of rods

Pupil masks

ε = 0.26

ε = 0.39

ε = 0.52

None

1.00

1.00

1.00

Illumination

0.87

0.81

0.75

Imaging

0.82

0.92

0.84

Both

0.74

0.68

0.50

Table 7

Rod counts for the images in Fig. 6. The manual counts are averages (and standard deviation) from 4 values by 2 different individuals.

Pupil masks

ε = 0.26, 41 cones

ε = 0.39, 43 cones

ε = 0.52, 50 cones

Automated

Manual

Automated

Manual

Automated

Manual

None

428

441 (4)

412

426 (5)

415

428 (6)

Illumination

477

489 (4)

533

549 (10)

499

507 (2)

Imaging

393

404 (5)

439

458 (8)

397

408 (4)

Both

452

461 (3)

523

530 (2)

508

515 (6)

Tables (7)

Table 1

Normalized autocorrelation central lobe FWHM changes due to pupil apodization corresponding to images in Fig. 4. Negative values indicate FWHM reduction that can be interpreted as sharpening of the image features.

Table 3

Cone counts for the images in Fig. 4. The manual counts are averages (and standard deviation) from 6 counts by 3 different observers.

ε = 0.26

ε = 0.39

ε = 0.52

Pupil masks

Automated

Manual

Automated

Manual

Automated

Manual

None

283

286 (1.3)

266

270 (1.5)

253

266 (2.1)

Illumination

306

309 (1.6)

318

322 (2.2)

380

385 (2.7)

Imaging

283

287 (2.3)

268

273 (1.3)

270

276 (2.6)

Both

301

306 (1.5)

323

329 (1.8)

350

354 (1.9)

Table 4

Normalized autocorrelation central lobe FWHM changes due to pupil apodization corresponding to images in Fig. 6. Negative values indicate FWHM reduction, that can be interpreted as sharpening of the image features.

Pupil masks

ε = 0.26 (%)

ε = 0.39 (%)

ε = 0.52 (%)

None

0.00

0.00

0.00

Illumination

−8.0

−12.7

−7.4

Imaging

−3.7

−2.2

3.3

Both

−11.2

−10.5

3.1

Table 5

Measured relative average photoreceptor intensities for images collected with constant PMT gain compared against predicted values (in parenthesis) corresponding to Fig. 6. The values used to describe the SCE are the best fit to our experimental data (ρcones = 0.15 mm−2 and ρrods = 0.11 mm−2) assuming a Gaussian intensity profile at the illumination pupil (σ = 2.9 mm) and a one Airy disk confocal aperture.

ε = 0.26

ε = 0.39

ε = 0.52

Pupil masks

Cones

Rods

Cones

Rods

Cones

Rods

None

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

1.00 (1.00)

Illumination

0.48 (0.68)

0.55 (0.73)

0.46 (0.42)

0.57 (0.49)

0.15 (0.21)

0.20 (0.27)

Imaging

0.59 (0.59)

0.72 (0.64)

0.33 (0.30)

0.36 (0.37)

0.11 (0.12)

0.13 (0.17)

Both

0.26 (0.40)

0.35 (0.47)

0.15 (0.13)

0.22 (0.18)

0.02 (0.03)

0.04 (0.05)

Table 6

Ratios of the average image intensity from cones to that of rods

Pupil masks

ε = 0.26

ε = 0.39

ε = 0.52

None

1.00

1.00

1.00

Illumination

0.87

0.81

0.75

Imaging

0.82

0.92

0.84

Both

0.74

0.68

0.50

Table 7

Rod counts for the images in Fig. 6. The manual counts are averages (and standard deviation) from 4 values by 2 different individuals.